Project Summary/Abstract Fats or lipids are thought to be deleterious to human health, but recent research has begun to identify beneficial roles for these molecules. Branched fatty acid esters of hydroxy fatty acids, named FAHFAs, are a newly discovered group of lipids that are produced in mammalian tissues (mouse and human), with highest levels in adipose (fat) tissue. Administration of purified FAHFAs to mice reverses diabetes and reduces inflammation, indicating that raising the levels of these lipids in vivo would be of therapeutic benefit. Indeed, analysis of FAHFA levels in humans revealed that people with type 2 diabetes had lower FAHFA levels than healthy individuals. Administration of pure FAHFAs in mice provides an experimental framework to study these lipids, but lipids make inferior drugs because they are not absorbed well and at high doses they can cause problems in the gastrointestinal tract. Therefore, an alternative approach to raising FAHFA levels would be to inhibit the proteins that are responsible for degrading these lipids in vivo. By blocking these proteins, FAHFA levels should increase leading to improved health, such as reduced inflammation. As new lipids, very little is known about the proteins and metabolic pathways (i.e., the biochemistry) that are present in the body to control the concentrations of FAHFAs. Recent work reported the first two enzymes capable of degrading these lipids, but these enzymes are not thought to be present in adipose tissue where the highest levels of FAHFAs are found. In new data introduced in this application, an adipose tissue enzyme called Ces3 in mice and CES1 in humans is found to degrade FAHFAs. And mice that lack Ces3 have higher FAHFA levels suggesting that this enzyme can also control FAHFA concentrations in a living animal. Ces3/CES1 are already interesting genes because the loss of these proteins from mice dramatically improves their metabolic health. As a result, Ces3/CES1 is a potential target for treatment of metabolic disease, but to date, no natural substrates for this enzyme have been reported. By defining the lipid substrates of Ces3/CES1 in vitro, in vivo, and pharmacologically, this application has the potential to provide the mechanistic insight necessary to promote the development of Ces3/CES1 inhibitors as a new class of anti-diabetic and anti- inflammatory drugs.